Acquired Immune Deficiency Syndrome (AIDS), AIDS related complex (ARC) and pre-AIDS are thought to be caused by a retrovirus, the Human Immunodeficiency Virus (HIV). The first AIDS related virus, HIV-1 (also known as HTLV-III, LAV-1 and ARV) has been well characterized. Another pathogenic human retrovirus named HIV-2 (formerly LAV-2) has now been isolated from West African patients with AIDS. See, e.g., WO 87/04459. HIV-2 has recently been shown (Guyader et al. Nature 326 662-669, 1987) to share a number of conserved sequences with HIV-1 and the Simian Immunodeficiency Viruses (SIV).
Even though other numbering systems are used in the art, for ease of understanding and comparison we have adopted herein the amino acid numbering system of Ratner et al., Nature, 313, 277-284, 1985 for the HIV-1 proteins and that of Guyader et al., Nature 326, 662-669 (1987) for the HIV-2 proteins. The amino acids in the peptides of this invention are designated by the single letter code as follows: ala=A, arg=R, asn=N, asp=D, cys=C, gln=Q, glu=E, gly=G, his=H, ile=I, leu=L, lys=K, met=M, phe=F, pro=P, ser=S, thr=T, trp=W, tyr=Y and val=V.
The initial immunodiagnostic tests for the detection of antibodies in the serum of patients infected with HIV-1 utilized the whole virus as antigen. Second generation tests made use of polypeptide sequences obtained by the recombinant DNA methodology. Cabradilla et al. Bio/Technology 4 128-133 (1985) and Chang et al. Bio/Technology 3, 905-909 (1985), for example, refer to bacterially synthesized viral protein fragments of 82 and 102 amino acid residues, respectively. EPA 202314 and 114243 refer to recombinant polypeptides spanning regions of gp41 and gp120 that are immunoreactive alone or in mixtures. Shoeman et al., Anal. Biochem. 161, 370-379 (1987) refers to several polypeptides from gp41 that are immunoreactive with antibodies present in sera from patients infected with HIV-1. None of the above assay procedures is, however, totally acceptable. Their lack of sensitivity is a critical shortcoming. It may permit blood containing virus to escape detection and thereby potentially result in the infection of blood product receivers and continued infectivity by undiagnosed AIDS carriers. Their lack of specificity (false positives) is also a problem--healthy individuals are told they may have AIDS. Such false positive may be caused by impurities. They may also be caused by shared epitopes with viruses unrelated to AIDS present in these antigen preparations. In this regard, Gallaher, Cell 50 327-328, 1987 has reported that a region of gp41 of HIV-1 shares a sequence of five adjacent amino acid residues with the respiratory syncytial virus and of four equally distributed amino acids of the measles virus F1 glycoprotein. Thus, even highly purified recombinant polypeptides containing this region, or any other common regions yet to be discovered, could potentially be responsible for false positives and the attendant unacceptable specificity. Finally, these prior art assays do not permit detection of very low levels of HIV antibodies. This disadvantages the assays in terms of their ability to detect AIDS infections at a very early stage, thereby delaying the start of treatment and permitting the possible spread of infection by blood samples and other body fluids before effective detection of AIDS infection.
In an attempt to solve these problems, diagnostic means and methods employing shorter HIV antigens are now being developed. Empirical methods to identify peptide sequences corresponding to unique and highly conserved epitopes of the HIV viruses are also now available. These methods are, for example, capable of assisting in the selection of short amino acid sequences which are more likely to be exposed on the surface of the native protein and thus useful as assay tools (for a review see Hopp and Woods, J. Immunol. Met. 88, 1-18, 1986). Although somewhat useful, these methods are no more than indicative. Nonetheless, they have been applied to identify epitopes present on the surface of viruses responsible for AIDS. For example, U.S. Pat. No. 4,629,783, International Patent Appl. PCT/US86/00831 and EPA 303224 refer to various synthetic peptides from the p18, p25, gp41 and gp120 proteins. These peptides are advantaged by the relative ease and lower cost with which they can be prepared and more importantly because of the reduced risk of obtaining false positives with them due to impurities or the presence of shared epitopes with viral proteins not related to AIDS.
While these smaller peptides are advantaged in terms of specificity over the earlier recombinant polypeptide and whole virus approaches to the diagnosis of AIDS infections, they have been less than satisfactory in terms of overall sensitivity, perhaps because the synthesized epitope is not able to assume and maintain a conformation that is recognized by the AIDS antibodies. Although the number of serum samples tested in each of these cases is very limited, specificity (few if any false positives) was found to be very high (95%-100%) with the small synthetic peptides but the overall sensitivity varied between 80% and 100%. In fact, in the only example where 100% sensitivity was attained only ten samples were tested. For example, Smith et al., J. Clin. Microbiol. 25 1498-1504, 1987 refers to two overlapping peptides, E32 and E34, that are highly immunoreactive. No false positives, out of 240 seronegative specimens, were obtained but the peptides missed three seropositive samples out of 322 (sensitivity of 99.1%). Wang et al. (Proc. Natl. Acad. Sci 83, 6159-6163, 1986) refers to a series of overlapping peptides (including amino acid residues of Smith's E32 and E34 peptides) among which one 21-mer peptide showed 100% specificity and 98% sensitivity (out of 228 seropositive samples taken from patients with AIDS, 224 were found positive with this peptide). And U.S. patent application 120,027, filed Nov. 13, 1987 refers to a short synthetic peptide spanning residues 606 to 620 (SGKLICTTAVPWNAS) of gp41 (HIV-1). This peptide is said to be immunoreactive with antibodies of patients infected by the AIDS viruses. The specificity was also excellent (63/63) but 6 seropositive specimens out of 57 confirmed positive could not be detected (sensitivity of 89%).
Gnann et al. (J. Virol. 61, 2639-2641, 1987 and J. Infec. Dis 156, 261-267, 1987) also refer to a series of overlapping peptides from a suspected immunodominant region of gp41 (HIV-1). Gnann et al. concluded that cys-605 was essential for the immunoreactivity of that segment of the gp41-(HIV-1) protein. They reported that a peptide having the sequence SGKLIC (606-611) was not immunoreactive with any of the 22 HIV-1 positive sera tested, while the addition of the cysteine residue to the N-terminus restored some immunoreactivity, 21 of 44 sera reacted with the 7-mer peptide (48% sensitivity).
Gnann et al. (J. Virol) also speculated that the cysteine residues at positions 605 and 611 of gp41 (HIV-1) might play a role in the antigenic conformation of this region perhaps by the formation of a cyclic structure via disulfide bonding. However, Gnann et al. never demonstrated that they did have a synthetic peptide wherein the two cysteine groups were linked by disulfide bonds.
Although Gnann et. al refers to peptides which are useful in identifying HIV-1 antibodies, even its peptides lack 100% sensitivity. For example, Gnann et al. (J. Virol. 61, 2639-2641, (1987)) report that while their 600-611 amino acid sequence detected 22 out of 22 positive sera, they also reported that similar tests carried out at the Centers for Disease Control, Atlanta, Ga. with the same 12-amino acid sequence (600-611) missed 1 out of 79 positive sera. And Gnann et al. in J. Infect. Dis. 156, 261-267, 1987 reported that the same 12-amino acid sequence was reactive with 131 out of 132 HIV-1 infected patients from the United States.
Gnann et al. Science 237, 1346-1349, 1987 reports a short linear synthetic peptide spanning residues 592 to 603 of gp42 (HIV-2) that contains two cysteines in a region homologous to the 605-611 region of gp41(HIV-1). This peptide reacted with 5 out of 5 sera taken from HIV-2 infected patients.
Other peptides containing amino acids 605-611 of gp41 of (HIV-1) are also referred to in the art. WO 86/06414 refers to peptide X(39), which is encoded by the region from about bp 7516 through bp 7593, and peptide XIII(79) which is encoded by the region extending from about bp 7543 through bp 7593, both containing the 7-amino acid sequence 605-611. These peptides are reported to be linear and no formation of cyclic structures is suggested. WO 87/06005 reports that a series of synthetic peptides encompassing the Cys(605)-Cys(611) residues of the HIV-1 envelope glycoprotein (gp41) undergo a series of spontaneous oxidative transformations upon solubilization in neutral or basic aqueous buffer. It speculates that as a result, the peptides when used in ELISAs are a random mixture of linear monomer, cyclic monomer, linear or cyclic dimers and linear polymers of various lengths. The application did not actualy demonstrate the presence of cyclic components and did not characterize the other various dimers and polymers possibly present. Moreover, it speculates that the polymer forms are the most important components for ELISA reactivity.
In addition to perhaps being complex mixtures of various oxidative forms of the peptide, the prior art peptides referred to above do not permit as early detection of AIDS infection as would be desirable. For example, Gnann et al. (J. Virol) reports that when the HIV-1 positive sera are diluted by a factor exceeding 500, some of these diluted sera are found to be negative thus indicating a low sensitivity of the peptide for early HIV detection.
These problems have been addressed by employing peptides that have been chemically cyclized to form a disulfide bridge between the relevant cysteines. E.g., M. Lacroix et al., Comparative Performance of Cyclic Versus Linear Peptides In An ELISA For HIV-1 And HIV-2 Specific Antibodies, No. 3147, June 1989 AIDS Conference, Montreal, Canada; and WO 89/03344. This invention is directed to improvements in such cyclic peptides.